Fused in Sarcoma (FUS) is a ubiquitous multifunctional RNA-binding protein (RBP) located in the nucleus. The abnormal and pathogenic aggregation of FUS in the cytoplasm of neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), termed FTLD-FUS or ALS-FUS. ALS- FUS cases are caused by mutations in the FUS gene. In these cases, the accumulation of FUS is thought to be driven by long-term increases in cytoplasmic FUS caused by mutations that decrease nuclear import through disruption of a conserved nuclear localization signal (NLS). However, it is unknown why FUS accumulates in FTLD-FUS cases. Moreover, it is unclear what causes cytoplasmic FUS in ALS to aggregate and become insoluble. We have discovered a novel mechanism that may explain both phenomena. We find that FUS can be phosphorylated and this event causes the cytoplasmic redistribution of FUS in multiple cells including human astrocytes and neurons. In particular, we find the DNA-damage, caused by chemical toxins, is a potent inducer of FUS phosphorylation. Furthermore, DNA-damage also causes cytoplasmic accumulation of EWS, TAF15, and TRN, which mimics a unique aspect of FTLD-FUS pathology. Preliminary evidence suggests that phosphorylation of FUS leads to increased amounts of FUS in the cytoplasm by disrupting a novel nuclear localization signal in the N-terminus. Consistent with this mechanism, we find that a FUS phospho-mimetic accumulates in the cytoplasm and forms aggregates. These aggregates co-label with markers of stress granules (SGs), RNA/protein granules that have been linked to the formation of inclusions in ALS-FUS and other forms of neurodegeneration. We theorize that cytoplasmic phosphorylated FUS can cause disease through a toxic gain of function by inducing granules that sequester RNA and RNA-binding proteins, impeding normal function. Importantly, we find that phosphorylated FUS occurs in the biochemically insoluble fraction of brains of human and mice with FUS inclusions. Finally, we find a large increase in ?-H2AX, a marker of DNA damage, in FTLD-FUS brains, supporting the idea that DNA damage and phosphorylation of FUS is a key component of disease pathogenesis. In this proposal we focus on the hypothesis that double-strand DNA damage induces phosphorylation of FUS by the DNA-dependent protein kinase (DNA-PK) causing FUS accumulation in the cytoplasm by impairing nuclear import. We will test this hypothesis by 1) Defining the kinase and types of DNA damage responsible for FUS phosphorylation, 2) Determining how phosphorylation of FUS causes cytoplasmic translocation and affects function, and 3) Determining the role of FUS phosphorylation in neurodegeneration. This research will provide insight into how FUS accumulation causes neurodegeneration and inform drug development strategies for ALS and FTLD. Our data suggest that methods to prevent FUS from forming pathogenic RNA/stress granules, potentially through modulation of the DNA- repair pathway or DNA-PK, may yield treatments for these devastating neurodegenerative diseases.